Role Of Cripto-1 In Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma(NPC)is a type of head and neck cancer,which is common in Southern China and Southeast Asia,especially in those of Cantonese origin.NPC carcinogenesis is a multi-step process involving many possible etiological factors.It is widely accepted that Epstein-Barr virus(EBV)infection, environmental factors and genetic susceptibility are the major factors involved.The epidemiological study of NPC showed a tendency of family aggregation,which suggests genetic susceptibility might play a pivotal role in the pathogenesis of NPC. However,the molecular mechanisms underlying NPC pathogenesis remain unclear. In addition,NPC tends to be early invasive and metastatic.Due to its originating form a hidden anatomical site,it is usually in the advanced clinical stage at the time of diagnosis.Hence,the prognosis for NPC is poor with a 5-year survival rate of less than 60%.Under such circumstances,it is of great clinical values to further understand the molecular mechanisms of this cancer and find valuable early diagnostic markers as well as novel therapeutic strategies.Human Cripto-1,also known as teratocarcinoma-derived growth factor-1 (TDGF-1),is a member of the Epidermal growth factor-cripto FRL1 cryptic (EGF-CFC)family(Cripto in humans,FRL1 in Xenopus,and Cryptic in mice), which is indispensable for early embryonic development.In vivo,this 188-amino acid glycoprotein,Cripto-1 has two activity patterns:as a cell surface co-receptor anchored by glycosylphosphatidylinositol and as a soluble protein after cleavage of the glycosylphosphatidylinositol linkage.Although identified as a marker for embryonic stem cells and generally absent from adult tissues,Cripto-1 is overexpressed in 75-80%of human breast,colon,and lung cancers,as well as 50-60%of testicular,stomach,pancreatic,and ovarian cancers.Furthermore, Cripto-1 expression is significantly increased in premalignant lesions,such as colon adenomas,intestinal metaplasia of the gastric mucosa and ductal carcinoma in situ (DCIS)of the breast.Recently,it has been reported that plasma Cripto-1 might represent a novel biomarker for the early detection of breast and colon carcinomas.In vitro and transgenic mice studies have shown that cripto-1 play an important oncogenic role during tumorigenesis by promoting cell proliferation,survival, migration and invasion,as well as inducing epithelial-to-mesenchymal transition(EMT),transformation,branching morphogenesis and tumour angiogenesis. Since high expression of Cripto-1 can be detected in human cancers,as compared to normal tissues,this might represent a new target for cancer therapy.Cripto-1 antisense oligonucleotides and neutralizing antibodies against Cripto-1 were able to significantly inhibit the in vitro and in vivo growth of human breast,colon,ovarian, testicular carcinoma cells and leukaemia cells.Until now,there is never any evidence that has shown a relationship between Cripto-1 expression and carcinogenesis of NPC.Based on the research review of Cripto-1 and its potential significance in the molecular mechnisms of NPC,the identification of its expression characteristics in NPC tissues and cell lines will help understand the mechanisms by which Cripto-1 contributes to the origin and progression of NPC.Furthermore,the implication of the role of Cripto-1 in NPC might provide new insight into understanding the molecular mechanism involved in NPC carcinogenesis and progression,and may lead to the development of new approaches for effective diagnosis and therapy.In this study,we performed the following researches to elucidate the characteristics and regulatory mechanisms of Cripto-1:CONTENTS AND METHODS1.Identification of expression characteristics of Cripto-1 in NPC and its significanceThe expression of Cripto-1 mRNA in 6 NPC cell lines(CNE1,CNE2,SUNE1, HNE1,C666-1 and HONE1)and 17 NPC biopsy specimens was detected by real-time reverse polymerase chain reaction(RT-PCR)and RT-PCR respectively.The expression of Cripto-1 protein in 6 NPC cell lines and 37 NPC tissues was detected by Western blot and immunohistochemistry(IHC)respectively.2.Effects of Cripto-1 knock-down on biological behaviors of CNE2 cellsSpecific anti-Cripto-1 short hairpin RNA was cloned into the downstream of human H1 polⅢpromoter in a pLVTHM vector containing EF1-GFP.NPC cell line CNE2 was transfected with the vector pLVTHM/shCripto-1 and then subjected to fluorescence activated cell sorter(FACS)analysis for GFP expression.A cell subclone CNE2-p1VTHM/shCripto-l was picked out,with stable knock-down of Cripto-1.Quantitative real time PCR was used to detect the efficiency of interference of cell clone,followed by Western blot method to detect the modulation of Cripto-1 protein.After the inhibition of the endogenous Cripto-1,the growth,cell cycle and invasion of cells were detected by MTT,plate colony formation,FACS and Boyden chamber assay in vitro respectively.3.Effects of Cripto-1 over-expression on biological behaviors of CNE1 cells Eukaryotic expression vector of Cripto-1,pCI/CR-1,was a gift from Prof. Caterina Bianco and Prof.David S.Salomon of NIH.After identification by restriction endonuclease analysis,PCR and DNA sequencing,the pCI/CR-1 plasmid was transfected into CNE1 cells,followed by G418 selection.Quantitative real time PCR was used to detect the efficiency of over-expression of cell clone.Western blot was used to examine the change of Cripto-1 protein.After the upregulation of the expression Cripto-1,the growth,cell cycle and invasion of cells were detected by MTT,plate colony formation,FACS and Boyden chamber assay in vitro respectively.4.Comparative proteomic analysis between CNEl/neo and CNE1/CR-1~+ cell lines with two-dimensional differential in-gel electrophoresis(2D-DIGE)To better understand the mechanism underlying the role of Cripto-1 and to search its potential signaling molecules,differential proteome analysis on two CNE1 cell lines with high and low Cripto-1 expression,CNE1/CR-1~+ and CNEl/neo,was conducted with 2D-DIGE.Differentially expressed protein spots were acquired by image analysis and identified by MALDI-TOF MS analysis.Semi-quantitative RT-PCR analysis were used to further verify candidate proteins in order to ensure the reliabilty of the proteome results.RESULTS1.Identification of expression characteristics of Cripto-1 in NPC tissues and cell linesQuantitive real-time PCR was used to detect the expression of Cripto-1 in NPC-derived cell lines(SUNE1,HNE1,CNE1,CNE2,HONE1 and C666-1)and immortalized nasopharyngeal epithelial cells NP69.The results of one way ANOVA analysis showed that the expression of Cripto-1 in 7 cell lines was significantly different from each other(F=48.783,P＜0.001).And compared with NP69 cells,the expression levels of Cripto-1 mRNA(P＜0.05)were significantly up-regulated in all 6 NPC cell lines.Western blot showed the expression levels of Cripto-1 protein were consistent with the expression levels of mRNA.SNK analysis showed that Cripto-1 mRNA was highly expressed in CNE2(ΔΔCt=-11.680±1.074)and C666-l(ΔΔCt=-10.637±0.637)with high metastatic ability,lowly expressed in CNE1(ΔΔCt=-3.523±0.648)and HNEl(ΔΔCt=-3.443±1.694)with no metastatic ability,and moderately in SUNEl(ΔΔCt=-6.447±0.396)and HONEl(ΔΔCt=-6.150±1.536).The expression levels of Cripto-1 mRNA were also examined in 17 NPC tissue samples,7 chronic nasopharyngitis tissue samples by semiquantitative RT-PCR analysis.Cripto-1 was higher expressed in 13 of 17 (76.5%)carcinomatous tissues compared with 2 of 7(28.6%)inflammatory tissues (P=0.042).In an immunohistochemical study,Cripto-1 staining was mostly observed in the cytoplasmic membrane and cytoplasm of carcinoma cells.No specific Cripto-1 staining was observed in normal adjacent nasopharyngeal epithelial cells and stroma cells in surrounding tissues.Cripto-1 was found to express in 54.1%(20/37)cases of NPC,higher then 20%(4/20)cases of chronic nasopharyngitis tissue samples(X~2= 6.176,P=0.013).The relationship between clinicopathological features and Cripto-1 expression in NPC was analyzed with Fisher's exact test.No significant associations were found between Cripto-1 expression and age,gender and T classification of NPC patients(P＞0.05).Interestingly,we observed that Cripto-1 expression was positively correlated with N classification(P=0.034),distant metastasis(M classification,P=0.036)and clinical stage(P=0.007)of NPC patients.With the evolvement of NM stage and clinical stage,the positive rate of Cripto-1 expression in NPC tissues increased.2.Effects of Cripto-1 knock-down on biological behaviors of CNE2 cellsPCR and DNA sequencing demonstrated that the lentivirus RNAi vector of (pLVTHM/shCripto-1)producing Cripto-1 shRNA was constructed successfully. The titer of concentrated virus was 4.2×10~5 TU/ml.CNE2 was transfected with recombinant lentivirus of small interference RNA targeting Cripto-1 and the negative control lentivirus(GFP~+/pLVTHM-lentivirus).After a selection of GFP expression by FACS,quantitative real time PCR and Western blot assays were used to detect the efficiency of RNAi targeting Cripto-1.CNE2/GFP~+/Cripto-l~-was established,with stable knock-down of Cripto-1.CNE2/GFP~+/Cripto-l~-cells showed a significantly reduced proliferation compared with CNE2 and CNE2/GFP~+/pLVTHM cells as determined by in vitro MTT assay(F=32.364,P＜0.001).In addition,CNE2/GFP~+/Cripto-l~-cells had a significant reduction in their ability to form colonies in plate as compared with CNE2 and CNE2/GFP~+/pLVTHM(F=25.475,P=0.001).However,the cell cycle distribution detected by flow cytometry has no significant differences between each other.These results indicated knock-down of Cripto-1 could reduce proliferation of CNE2 cells.The results of in vitro invasion assay showed that CNE2/GFP~+/Cripto-1~-cells had significantly reduced invasiveness as compared with CNE2 and CNE2/GFP~+/pLVTHM cells(F=153.75,P＜0.001).These results showed knock-down of Cripto-1 results in a downregulated invasion of CNE2 cells.3.Effects of Cripto-1 over-expression on biological behaviors of CNE1 cellsAfter identification by restriction endonuclease analysis,PCR and DNA sequencing,the pCI/CR-1 plasmid was transfected into CNE1 cells,followed by G418 selection.Successful overexpression of Cripto-1 was confirmed by quantitative real time PCR and Western blot assays.Finally,CNE1/CR-1~+ with stable over-expression of Cripto-1 was established.CNE1/CR-1~+ cells showed a significantly increased proliferation compared with CNE1 and CNEl/neo cells as determined by in vitro MTT assay(F=49.070, P＜0.001).In addition,CNE1/CR-1~+cells had increased ability of colony formation in plate significantly as compared with CNE1 and CNEl/neo(F=5.861,P=0.039). Besides,compared with CNEl/neo cells,CNE1/CR-1~+cells showed a significantly increased number of cells in S period(t=12.441,P＜0.001)and a significantly decreased in G0/G1 period(t=-5.083,P=0.007)by flow cytometry.These results indicated over-expression of Cripto-1 promoted the proliferation of CNE1 cells.The results of in vitro invasion assay showed that CNE1/CR-1~+ cells had significantly increased invasiveness as compared with CNE1 and CNEl/neo cells (F=106.081,P＜0.001).These results showed over-expression of Cripto-1 leads to an upregulated invasion of CNE2 cells.4.Comparative proteomic analysis between CNEl/neo and CNE1/CR-1~+ cell lines with two-dimensional difference gel electrophoresis(DIGE)By 2D-DIGE image analysis,the expression levels of 49 protein spots were found quantitatively changed(more than 2 fold up or down quantitatively)between CNEl/neo and CNE1/CR-1~+cells,among them the expression levels of 37 protein spots were increased and the expression levels of 12 protein spots were decreased in CNE1/CR-1~+ cells.23 proteins were identified successfully by MALDI-TOF MS analysis,with 17 upregulated and 6 downregulated.DJ-1 and 1433G may have a close association with the activation of PI3K-Akt and MAPK signaling pathway by Cripto-1 in NPC.ANXA3 may have a close association with the role of Cripto-1 in metastasis of NPC. Conclusions1.The expression of Cripto-1 is upregulated in NPC and correlates statistically with the malignant status of NPC.2.Cripto-1 promots the growth and invasion of NPC cells in vitro and might play a pivotal role in the tumorigenesis and progression of NPC.3.Differentially expressed proteins DJ-1,1433G and ANXA3,got from 2D-DIGE analysis,may be involved in the role of Cripto-1 in NPC.